Capillary gel electrophoresis is one of the most widely used separation techniques in the biologically-related sciences. Molecular species such as proteins, peptides, nucleic acids, and oligonucleotides are separated by causing the species to migrate in a buffer solution under the influence of an electric field. The buffer solution normally is used in conjunction with a low to moderate concentration of an appropriate gelling agent such as agarose or polyacrylamide to minimize the occurrence of mixing of the species being separated. Two primary separating mechanisms exist: a) separations based on differences in the effective charge of the species; and b) separations based on molecular size.
The first of these mechanisms is generally limited to low or moderate molecular weight materials, such as small oligonucleotides (about 1 to about 50 nucleotides in length). This is because there is typically an insignificant difference between the effective charges of high molecular weight materials, making the task of separation difficult or impossible.
Separations based on molecular size are generally referred to as molecular "sieving". Molecular sieving utilizes gel matrices having controlled pore sizes as the separating medium. The separation results from the relative abilities of the different size molecular species to penetrate through the gel matrix; smaller molecules move more quickly than larger molecules through a gel of a given pore size.
Medium-to-high molecular weight oligonucleotides (greater than about 50 nucleotides in lenght), polypeptides, and proteins are commonly separated by molecular sieving electrophoresis. Proteins are heteropolyelectrolitic (i.e. an approximate equivalent number of negative charged and positive charged moieties where the overall molecule has a net neutral charge). As such, proteins become charged molecules as they transit a charged capillay column. Accordingly, in order to separate proteinaceous materials based upon the size of the molecules, these materials must have the same effective charge to mass ratio as they traverse the capillary column.
Achieving the same effective charge to mass ratio is commonly accomplished by treating the proteinaceous materials with a surfactant, such as sodium dodecyl sulphate ("SDS"), and utilizing a polyacrylamide gel material as the seiving medium. Such a procedure is referred to as sodium dodecyl sulphate polyacrylamide gel electrophoresis ("SDS-PAGE"). See, for example, Gel Electrophoresis of Proteins: A Practiced Approach (Second Ed). B. D. Harnes & D. Rickwood, Eds. IRL Press, Oxford University Press, 1990. See also, New Directions in Electrochoretic Methods. T. W. Jorgenson & M. Phillips, Eds. published by American Chemical Society, Washington, D.C. 1987. Both of these references are incorporated fully herein by reference.
A surfactant, such as SDS, comprises a hydrophobic (water-hating) "tail" and a hydrophillic (water-loving) "head." Thus, a surfactant interacts with a protein species via hydrophobic interactions between the hydrophobic "tail" of the surfactant and the protein species. Upon ionization, the hydrophillic "head" of the surfactant molecules surrounding the protein species become negatively charged, positively charged, or remain neutral; upon ionization, SDS becomes negatively charged. Accordingly, an SDS:protein complex has a uniform charge distribution, and such a complex can then be separated based upon size relative to the pore-size distribution throughout the gel matrix.
Commercially available capillary electrophoresis instruments the P/ACE.TM. high performance capillary electrophoresis system (Beckman Instruments, Inc., Fullerton, Calif., U.S.A.), utilize a detection system based upon ultra-violet ("UV") light absorption. While UV detection of SDS-protein complexes in polyacrylamide filled capillaries is possible, such detection is limited to a specific wavelength detection of about 250 nm and higher. This is because of the high UV absorbance associated with both crosslinked and uncrosslinked polyacrylamide gels.
Such dection limitations are a distinct disadvantage particularly with respect to the analysis of proteins. This is because proteins absorb UV light very strongly at 214 nm, due to peptide bonds within proteins. Thus, UV detection of proteins should be conducted at about 214 nm. However, because of the 250 nm and higher detection limitations created by the use of polyacrylamide gels, the sensitivity and selectivity of UV detection of proteinaceous materials using polyacrylamide-based gel systems is limited.
Accordingly, UV detection of surfactant:proteinaceous materials would be greatly improved if on-column detection was conducted at lower UV wavelengths. This, in light of the foregoing, requires molecular sieving materials that do not suffer the drawbacks of polyacrylamide gels.